(a) Field of the Invention
This invention relates to a method for purifying proteins. More particularly, this invention relates to a high-recovery, high-resolution method of purifying anti-hemophilic factor VIIII:C ("AHF") by using column chromatography techniques in the presence of sugars, polyhydric alcohols, amino acids, or salts. The AHF preparations obtained by the present invention are also of high purity.
Factor VIII procoagulant protein (AHF) is a plasma protein that has the ability to correct the clotting defect in hemophilic plasma. In fact, the activity of AHF is measured by its ability to induce clotting in hemophilia A plasma.
One unit of AHF is the amount present in one milliliter of normal, adult male plasma. The standard used herein, a World Health Organization Standard, is available from the National Institute for Biological Standards and Control, Holly Hill, Hampstead, London NY3 6RD, England.
(b) Discussion of the Prior Art
The primary therapeutic use of AHF has been its intravenous administration to hemophilic patients. At first, this involved infusion of whole blood or fresh-frozen plasma, which required long infusion times and often caused hypervolemia.
Use of plasma cryoprecipitate ("cryo") still required long infusion times. The cryo was not completely soluble in the solvent used for infusion and required filtration. Its AHF content was low and highly variable. A large cryo volume was required, and assaying multiple sealed sterile containers for AHF content before use was very cumbersome and often omitted. Thus, the exact amount of cryo available for therapy could not be ascertained. Moreover, cryo required storage at -20.degree. C. in large plastic blood bags and, therefore, availability of large refrigeration facilities. This primitive method made at-home treatment impossible.
Use of lyophilized AHF concentrates solved many of the above problems. The concentrates are generally obtained by subjecting plasma to cryoprecipitation followed by a second precipitation with polyethylene glycol. The concentrates are stable under refrigeration; they dissolve completely in the reconstituting liquid, and may be reconstituted in lower volumes than cryo (10-30 ml) to give assayable concentrations of AHF 10-30 times higher than are present in whole plasma.
Unfortunately, the yield (AHF activity in the product/AHF activity in the starting plasma) obtained by use of this method is about 20%. This makes the cost of the factor excessive. In addition, the required infusion volume remains rather high (30 ml/1000 units of AHF), which again creates storage problems and makes at-home administration difficult, thus adding to the cost of therapy.
Furthermore, these concentrates contain less than 0.1% AHF protein and 99.9% contaminating proteins including specific blood-type antibodies that cause hemolysis, proteins that may cause immunologic abnormalities including an inversion of the T-cell ratio (helper/suppressor) which resembles AIDS, fibrinogen, fibronectin, von Willebrand factor and other proteins. Finally, the lyophilized concentrates are often contaminated with viruses (such as hepatitis B and non A/non B viruses or, possibly, viruses responsible for AIDS). Heating these preparations in the liquid state destroys most microorganisms, but also causes denaturation of a substantial portion of the AHF.
Chromatographic techniques (both ionic and hydrophobic chromatography) have been used, but only in the laboratory. Yield has been low (30-40% maximum) and resolution very poor.
Clearly, there is a need in the art for purer AHF preparations at a lower cost and a higher yield.
Attempts at satisfying this need include use of immunoaffinity chromatography using monoclonal antibodies: Zimmerman, et al, Characterization of the Human Factor VIII Procoagulant Protein with a Heterologous Precipitating Antibody, Proc. Natl. Acad. Sci. (U.S.A.) 79: 1648 (1982) and U.S. Pat. No. 4,361,509 (issued 11/30/82). Although these references report a 164,000-fold purification from plasma, the procedure involves six steps and the overall recovery is about 12% without a heating step.
J. J. Morgenthaler, Chromatography of Antihemophilic Factor on Diaminoalkane- and Aminoalkane-Derivatized sepharose, Thromb. Haemostas., (Stuttgart) 47(2): 124 (1982) discloses use of acetate-lysine buffer on modified sepharose columns to purify Factor VIII:C from polyethylene glycol precipitated AHF. The authors indicate that hydrophobic rather than ionic forces govern the behavior of these chromatographic columns, but the reference is silent on yield, or purity of the product.
Austen, D. E. G. and Smith, J. K., Factor VIII Fractionation on Aminohexyl Sepharose with Possible Reduction in Hepatitis B Antigen, Thromb. Haemostas. (Stuttgart) 48(1): 46 (1982), disclose a method of processing plasma by aminohexyl sepharose column chromatography using 9.times.150 mm columns, acetate-lysine washing buffer and a saline gradient for elution. The reported maximum yield was 46% and the purity about 100-fold over plasma. The primary benefit of the procedure is said to be the ability to process larger samples. In addition, the authors report indications that the procedure slightly decreases contamination with Hepatitis B virus (by about 1.5 orders of magnitude, based on particle content).
A. Faure, et al, Improved Buffer for the Chromatographic Separation of Factor VIII Coagulant, J. Chromatog. 257: 387 (1983), disclose use of 1 and 10% saccharose in acetate-lysine buffers to improve the yield of Factor VIII during chromatography of cryoprecipitate on aminohexyl sepharose. The only buffer reported to be consistently effective in increasing Factor VIII separation from protein and recovery is one containing about 1% (0.03M) saccharose and 1% albumin. According to the authors, sugar was added to inhibit the formation of a molecular complex between Factor VIII and activated Factor IX and Factor X; albumin was added to eliminate non-specific adsorption. In one experiment, this buffer almost doubled the recovery of Factor VIII coagulant from plasma (not cryoprecipitate) over that obtained using either acetate lysine buffer alone, or acetate-lysine buffer with sacharose. However, the reported increase in recovery is difficult to interpret because absolute recovery figures are not given, nor are purity or resolution data. Use of sucrose alone did not result in a consistent increase in yield.
Lundblad, R. L. et al, The Effect of Dextrose on Chromatography of Antihemophilic Factor (Factor VIII), Thrombosis Research, 1: 197 (Pergamon Press, Inc. 1972), disclose that addition of 0.50M dextrose in the eluting buffer of bovine Factor VIII ion exchange column chromatography (on TEAE-cellulose) slightly improves the purity of the product and increases the yield of the peak fractions from 15-45% to 60-70%. However, the resolution was slightly reduced.
Neither Lundblad nor Faure use higher concentrations of sugar. Lundblad suggested that sucrose might bind to the cellulose matrix and prevent nonspecific adsorption of the protein. Faure suggested that sucrose addition might prevent complex formation between AHF and factors IXa and X. No substantiating evidence was given for either suggestion.
Arakawa, T. and Timasheff, S. N., Stabilization of Proteins by Sugars, Biochem. 21: 6536 (1982) disclose that many sugars cause preferential hydration of proteins in aqueous systems and, hence, serve to stabilize proteins in such systems. The article states that in sugar solutions, the equilibrium shifts towards a more tightly folded conformation. However, the article does not involve Factor VIII, nor protein purification, nor column chromatography. The disclosure of this article is incorporated by reference.
A variety of other articles disclose that sugars, polyhdric alcohols, amino acids or salts serve to stabilize proteins in aqueous systems: Arakawa, T. and Timasheff, S. N., Preferential Interactions of Proteins with Solvent Components in Aqueous Amino Acid Solutions, Arch. Biochem. Biophys. 224(1): 169 (1983); Pittz, E. P. and Timasheff, S. N., Interaction of Ribonuclease A with Aqueous 2-methyl-2,4-pentanediol at pH 5.8, Biochem. 17(4): 615(1978); Gekko, K. and Timasheff, S. N., Mechanism of Protein Stabilization by Glycerol: Preferential Hydration in Glycerol-Water Mixtures, Biochem. 20: 4667 (1981); Lee, J. C. and Timasheff, S. N., The Stabilization of Proteins by Sucrose, J. Biol. Chem. 256(14): 7193 (1981); Gekko, K. and Morikawa, T., Preferentail Hydration of Bovine Serum Albumin in Polyhydric Alcohol-Water mixtures, J. Biochem. 90: 39-50 (1981); and Arakawa, T. and Timasheff, S. N., Preferential Interactions of Proteins with Salts in Concentrated Solutions, Biochem. 21: 6545-6552 (1982). Again none of these articles disclose anything about protein or AHF purification, or column chromatography. The disclosures of these articles are incorporated by reference, however, because they contain techniques and data for determining preferential hydration of proteins that are useful in the practice of this invention. Pertinent excerpts of these disclosures have also been specifically incorporated in this application for convenient reference.
Finally, sugars and polyhydric alcohols have been used to preserve enzymatic or other activity of proteins and stabilize their structure after the proteins have been isolated from their native media. This procedure has been used during heating and lyophilization after purification.
As used in this application the following terms shall have the meanings ascribed to them below:
"Biological fluid" means any solution or suspension medium which contains or can contain a protein without causing its permanent denaturation or inactivation, including without limitation: plasma, urine, culture media, buffers and physiological solutions.
"Hydration additive" means individually or collectively sugars, polyhydric alcohols, amino acids and salts use of which in protein purification by chromatography increases yield, purity or resolution of the protein so purified. The use of this term is for convenience. Although the present inventors have observed a close correlation between optimization of the present process at specific levels of preferential hydration and have used protein hydration data as a marker for the amount of hydration additive used in the present invention, it is not to be assumed that hydration of a protein and improvement in its purification by chromatography are necessarily related as cause and effect.